Introduction

The process of aging is marked by an intricate cascade of physiological, cellular, and molecular alterations. The process of senescence initially manifests as cellular senescence, cell cycle arrest, morphological alterations, and the emergence of the senescence-associated secretory phenotype (SASP). As individuals age, the accumulation of senescent cells gives rise to age-related pathological changes encompassing osteoporosis, brain diseases, neurodegeneration, vascular diseases, metabolic dysfunction, and physiological impairment1. Senescent cells actively secrete a diverse array of pro-inflammatory cytokines, lipids, and proteases. In the context of aging, unprogrammed cellular senescence results in the accumulation of senescent cells2. The SASP exerts paracrine effects on neighboring cells. In contrast to quiescent or apoptotic cells, senescent cells exhibit heightened metabolic activity and can be eliminated by immune cells. The gradual impairment of the immune system’s ability to eliminate senescent cells from tissues contributes to the development of age-related chronic diseases3. The characterization of cellular senescence is complex, and there lacks a single specific biomarker capable of definitively identifying senescent cells. Although cellular senescence is generally considered an irreversible process, there is a variety of evidence suggesting that senescent cells can re-enter the cell cycle. The p53/p21/p16/Rb pathway predominantly mediates cell cycle arrest in association with cellular senescence4. The investigation of anti-aging medications is currently underway with the aim of delaying or treating a range of age-related ailments. Some natural compounds, such as resveratrol, soy isoflavones, and rosmarinic acid, possess the ability to decelerate the aging process and prolong lifespan. Additionally, certain synthetic compounds like beta blockers, metformin, and renin-angiotensin-aldosterone system inhibitors have also been shown to have similar effects5,6. The maximum human lifespan is limited. Improvements in lifestyle and advancements in medical technology are the primary factors contributing to increased life expectancy. Environmental conditions have the potential to impact molecular mechanisms and potentially alter the cellular epigenome, thus influencing cell destiny.

Epigenetics is closely related to aging, and all cells in multicellular organisms have the same DNA. However, epigenetic mechanisms can activate different genes, causing genetically identical cells to adopt distinct fates. The gradual accumulation of epigenetic changes associated with aging may result in abnormal gene expression and disrupt cellular metabolic homeostasis. Changes in DNA methylation, histone post-translational modifications, chromatin organization, and remodeling can affect health span and longevity7. SMYD3 is a discovered protein that plays a role in histone methylation. It belongs to the methyltransferase SET and mynd domain families, with its expression being elevated in various types of cancer. It can lead to changes in the spatial structure of chromosomes, which alter the tightness and openness of the transcription complex. This regulation affects gene transcription, subsequently impacting oncogenes, cell cycle genes, and adhesion-related genes. It inhibits tumor cell apoptosis while promoting cell proliferation, invasion, and metastasis. In preliminary work, we confirmed that SMYD3 methylation can promote cellular senescence. Additionally, following virtual screening in the protein database, we successfully identified lead compounds and subsequently modified their structures via chemical synthesis. Through systematic modification and synthesis of these lead compounds, we developed and characterized a novel low-toxicity small molecule, designated ZYZ-384. This compound exhibits potent binding affinity for SMYD3 and effectively inhibits its enzymatic activity. Additionally, we have secured the relevant patents8. Building upon these findings, we aim to investigate whether ZYZ-384 possesses anti-aging pharmacological effects.

Material and method

Reagents and animal

Galactose (D8310), mouse IL-1β ELISA kit (SEKM-0002), mouse IL-6 ELISA kit (SEKM-0007), mouse MCP-1 ELISA kit (SEKM-0108), catalase (CAT) activity assay kit (BC0205), malondialdehyde (MDA) content assay kit (BC 0025) and senescence-associated β-galactosidase (SA-β-Gal) stain kit (G 1580) were bought from Solarbio (Beijing, China). BCA protein colorimetric assay kit (E-BC-K318-M) was got from Elabscience (Wuhan, China). Vascular endothelial cells derived from mouse (SVEC4-10) and human (HMEC-1) were received from National Collection of Authenticated Cell Cultures (Shanghai, China). MCDB 131 medium (10372019) and DMEM medium (11965092) were accepted from Gibco (Shanghai, China). Angiotensin II (Ang II) (GP10023) was acquired from GlpBio (Shanghai, China). in vivo-jetPEI (101000040) was gained from Univ-Bio (Shanghai, China). ELISA kit for superoxide dismutases (SOD) (MES134Mu) was earned from cloud-clone corp (Wuhan, China). Anti-CDKN2A/p16INK4a antibody (ab211542), anti-p 21 antibody (ab188224), anti-Rb antibody (ab181616), anti-Hsp 90 antibody (ab59459), anti-SMYD3 antibody (ab187149), anti-NF-κB p65 antibody (ab16502), Anti-Histone H 3 (tri methyl K4) antibody (ab213224) were gained from Abcam (Shanghai, China). Cell counting kit-8 (CCK-8) (C0038), BeyoClick™ EdU cell proliferation kit (C0071S), RIPA cell lysis solution (P0013B) and β-galactosidase staining (C0602) were purchased from Beyotime (Shanghai, China).

Cells culture

SVEC4-10 murine and HMEC-1 human vascular endothelial cells were maintained in DMEM and MCDB 131 media respectively, both containing 10% fetal bovine serum. Following medium replacement and PBS washing at 80% confluency, enzymatic dissociation with trypsin was initiated and halted immediately upon observable cell rounding. The harvested cells were subsequently transferred to a 37 °C incubator with a 5% CO2 atmosphere for continued expansion.

Cells viability assay

SVEC4-10 murine and HMEC-1 human endothelial cells were seeded in 96-well plates at 5 × 103 cells/well. Following 24 h stabilization, treatment groups were established through administration of ZYZ-384 at graded concentrations (0, 28.74, 57.47, 114.94, 172.41, 229.88, 287.35 µM). After 48-hour incubation under standard conditions (37 °C, 5% CO2), cellular viability was assessed by adding 10 µL of CCK-8 reagent to each well. After incubating for 4 h, the absorbance value was measured at 450 nm to determine the optimal drug concentration. The cells were re-inoculated at a density of 5 × 103 cells per well, and simultaneously treated with ZYZ-384 and Ang II at a concentration of 100 nmol/L in the medium for 48 h. Following incubation, 10 µL of CCK-8 solution was added to each well, and the cells were further incubated in the dark for 4 h. Subsequently, the absorbance value at 450 nm was measured using Thermo scientific microplate reader (Shanghai, China).

Senescence-associated β-galactosidase (SA-β-Gal) staining assay

The cells were cultured in a 6-well plate at a density of 1 × 105 cells per well. Following removal of the culture medium, the cells were washed once with phosphate buffer saline. Subsequently, each well was treated with 1mL of β-Gal fixing solution and incubated at room temperature for 15 min to soak the cells. After rinsing with phosphate buffer saline, the staining solution was prepared according to the instructions provided in the kit. 1 mL of dyeing solution was added to each well. The plate was incubated overnight at 37 °C and observed under an optical microscope.

EdU staining assay

The cells, numbering 5 × 103, were cultured on a glass cover slip and subsequently transferred to a 48-well plate. A staining solution of 10 µM 5-ethynyl-2’ -deoxyuridine was added to the wells, followed by incubation for a duration of 2 h. The culture medium was then removed from the plate and fixation was achieved using 200 µL of a 4% paraformaldehyde solution for a period of 15 min. Subsequently, the plate was washed with an appropriate washing liquid. Each well of the plate received click additive solution and underwent incubation away from light for a duration of 30 min. Finally, cell nuclear staining was performed using Hoechst 33,342 for a period of 10 min.

Immunofluorescence assay

Following medium removal, adherent cells were initially rinsed with PBS. All experimental groups underwent fixation with 4% paraformaldehyde (30 min), followed by 10-min permeabilization in 1% Triton X-100 and three PBS washing cycles. Subsequent blocking procedures employed goat serum-containing buffer for nonspecific site saturation. The cells were treated with p16 antibody (1:100), p21 antibody (1:100), and p53 antibody (1:100) and incubated overnight at 4 °C. After cells were triple washed with phosphate buffer saline, the goat anti-rabbit IgG antibody (1:1500) was added to the cells and incubated at room temperature in the dark for 1 h. The cover slip containing 4’, 6-diamidino-2- phenylindole covered the cells. The cell images were captured by a fluorescence microscope.

D-galactose animal model and natural aging model

Fifty C57 BL6/J mice, 8 weeks of age, were categorized into five groups. The control group included 10 mice, while the remaining 40 animals received subcutaneous injections of D-galactose at a dose of 100 mg/kg/day for a duration of two weeks. Forty modeling animals were further divided into the model group and ZYZ-384 gradient dose groups (2.5 mg/kg, 5 mg/kg, and 10 mg/kg). D-galactose was continuously administered while ZYZ-384 was orally administered. After a continuous administration period of six weeks, samples including animal plasma, heart, liver, spleen, kidney, and brain were collected.

Fifty C57BL6/J mice of 13 months age were divided into 5 groups. There were 10 subjects in the natural aging control group, and another 40 subjects in the ZYZ-384 gradient dose orally group (2.5, 5, and 10 mg/kg), as well as the siRNA SMYD3 group with tail vein intravenously injection of 200 µL. siRNA SMYD3 or nontargeting siRNA-NC was mixed with 5% glucose and 6.4 µL of polyethylenimine to form a stable compound of 200 µL, which was then administered intravenously. The intravenous injection is repeated every one week. After being administered continuously for 12 weeks, animal behavior was monitored and animal heart, spleen, liver, kidney, and brain were collected. During the rearing period, the animalsʼ diet and activities were unrestricted. All procedures were conducted in compliance with pertinent guidelines and regulations. Upon completion of the experiment, the animals were deeply anesthetized with nebulized isoflurane gas for euthanasia. All procedures were conducted in accordance with the reporting standards of the ARRIVE guidelines. The Animal Ethics Committee of Macau University of Science and Technology approved the experimental protocol (No. 20230184).

Animal ethology analysis

Rotating rod test

Utilize the rotating rod fatigue instrument (IITC755, Yuyan Instruments Co.,Ltd., Shanghai, China) to conduct the experiment. Set the run limit to 300 s and initialize the start rpm to 4 r/min. The maximum rotational speed was 40 r/min, and the ramp time was 30 s. After the mice were placed on the rotating rod for 5 min, the time from the start of rotation to the drop was recorded.

Running experiment

Utilizing an animal treadmill (DB0-30, ZHISHUDUOBAO Biological Technology Co., Ltd., Beijing, China), adjust the running duration to 30 min, set the electrical stimulation intensity at 0.8 mA, establish a stimulation frequency of 5 times per 8 s, and cease recording time when the mouse ceases running after three consecutive electrical stimulation.

Barnes maze experiment

The time taken by the mouse to reach the dark chamber was recorded using the Barnes Maze. Prior to the formal test, three training sessions were conducted with a 24-hour interval between each session. In case the mouse failed to find the dark chamber during the experiment, their longest duration of search was documented.

Hematoxylin-eosin staining

The heart, liver, spleen, kidney, and brain samples had a thickness ranging from approximately 0.2 to 0.3 cm, with dimensions of 1 cm × 1 cm × 0.5 cm. The sample was fixed in 10% neutral formaldehyde for a duration of 24 h. The tissue underwent a rinsing process with running water lasting for 2 h. Initially, the tissue samples were subjected to xylene infiltration, followed by immersion in alcohol solutions of increasing concentrations of 50%, 70%, 80%, 95% and 100% individually for a period of 30 min each. The paraffin embedded tissue was sliced and the thickness was 4 μm. The prepared slices were dried in a 60 °C drying oven for 2 h. The slices were dewaxed in xylene for a duration of 20 min. Subsequently, they were immersed in anhydrous ethanol for a period of 5 min. After that, the slices were consecutively submerged in ethanol solutions with concentrations of 90%, 80%, and 70% respectively, each time lasting for a duration of 2 min. The slices were stained with hematoxylin for 10 min and eosin for 30 s, followed by rinsing with 70%, 80%, 90% and 100% ethanol apiece for 10 s each. The slices were permeated with xylene for 5 min, and microscope observation was conducted after sealing them with neutral resin.

Immunohistochemical method

Each sample was dewaxed in xylene for 20 min, soaked in anhydrous ethanol for 5 min, and dipped in 90%, 80%, 70% ethanol for 2 min respectively. Then the slices were immersed in sodium citrate liquid for 10 min, blocked by immunol staining blocking buffer for 60 min, and incubated with p16 antibody (1:100), p21 antibody (1:100), Rb antibody (1:100), Hsp90 (1:100), SMYD3 (1:100), NF-kB p65 (1:100), H3K4 (1:100) for 24 h. The slices were rinsed 3 times with PBS. The II antibody were added (1: 200) and incubated with slices for 1 h and cleaned with PBS for 3 times again. Then the slices were mixed into DAB developer for image development, steeped hematoxylin for 5 min, soaked in tap water for 15 min, and dipped into 70%, 80%, 90% and 100% ethanol for 10 s respectively. In the end, xylene permeate the slices for 5 min. Microscope observation after sealing with neutral resin.

Enzyme-linked immunosorbent assay

Standard aperture, plasma sample aperture and control aperture were set up respectively. standard apertures were set and added 100 µL standard products of different concentrations in turn. 100 µL standard diluent was added to the blank well. The samples were added into the remaining apertures. 96-well plate was incubated at 37 °C for 1 h. 96-well plate was mixed into the test solution and incubated for 1 h. Discard the liquid in the hole, wash each hole with 350 µL scrubbing solution and tap the 96-well plate on the absorbent paper to remove all the liquid in the well. The plate was washed three times. The OD values of SOD, MDA, IL-1β, IL-6, MCP-1, ALT, AST, GLOB, CREA, CK, BUN, Cat, TC and TG of each sample were measured by microplate reader at 450 nm immediately after the suspension solution was added.

Metabolomics assay

High-resolution untargeted metabolomics analysis of lung samples from the control, model, and ZYZ-384 groups was performed. After slowly thawing the samples at 4 °C, an appropriate amount of samples was added to a pre-cooled methanol/acetonitrile/aqueous solution (2: 2: 1, v/v). The mixture was vibrated and ultrasonic concussion at low temperature for 30 min, followed by standing at −20 °C for 10 min. Subsequently, it was centrifuged at 14,000 g and 4 °C for 20 min. The supernatant was vacuum dried and reconstituted with 100 µL acetonitrile solution (acetonitrile: water = 1: 1, v/v) before being swirled and centrifuged again at 14,000 g and 4 °C for 15 min. Finally, the supernatant was taken for analysis. Metabolites in the samples were detected by the UHPLC-Q-TOF/MS method (AB SCIEX, Agilent, USA).

Frozen section

The freshly harvested tissues are rapidly immersed in liquid nitrogen for a duration of 15 min, followed by transfer to long-term storage at −80 °C. Prior to sample utilization, the cryo-cut microtome is adjusted to a temperature of −20 °C. The tissue is embedded in OCT reagent and affixed onto the cutting machine’s worktable using a circular plate. Thin sections with a thickness ranging from 10 to 15 μm are sliced from the sample utilizing the cryo-cut microtome. Subsequently, the tissue undergoes SA-β-Gal staining method as previously described and is observed under a conventional optical microscope.

Statistical analysis

The data analysis of experimental instances was conducted using SPSS 23.0 software, and the generation of visual outputs was achieved through GraphPad Prism 9.0. Non-parametric analysis was applied to trial data, with statistical significance set at P < 0.05.

Results

Analysis of the cell growth ability of ZYZ-384

The cells were cultured with compounds that were synthesized and patented in our prior research report8. After co-cultivating ZYZ-384 with human HMEC-1 or mouse SVEC4-10 cells for 48 h, we determined the optimal drug concentration of ZYZ-384. We determined that the optimal gradient concentration of ZYZ-384 in human HMEC-1 cells was 12.5 µM, 25 µM, and 50 µM, and the optimal gradient concentration of ZYZ-384 in mouse SVEC4-10 cells was 25 µM, 50 µM, and 100 µM (Fig. 1A and B). Angiotensin II (Ang II) can activate angiotensin receptors to accelerate biological aging, leading to senescence-associated secretory phenotype (SASP) and chronic inflammation9. Therefore, we used Ang II as an inducer of cell senescence to induce cellular aging. The proliferation activity of human HMEC-1 or mouse SVEC4-10 cells was significantly diminished upon the addition of Ang II, as compared to the control group. The proliferation activity of both cell types showed a varying degree of recovery with the increase in ZYZ-384 concentration (Fig. 1C and D). We know that positive expression of SA-β-gal indicates cell aging10. We utilized SA-β-Gal staining to re-stain the two cell types in order to observe the proliferation impact of ZYZ-384 on these cells. When Ang II was added to the cells, the number of cells showing a blue reaction compared to the control group without Ang II increased significantly, indicating an increase in the number of aging cells. As ZYZ-384 was cultured with cells at increasing concentrations, the number of cells exhibiting a blue reaction decreased significantly, indicating a reduced presence of senescent cells (Fig. 1E). The overall results suggest that ZYZ-384 has a promoting effect on cell proliferation.

Fig. 1
figure 1

The effects of ZYZ-384 on the inhibition and growth rate of human HMEC-1 or mouse SVEC4-10 cells. The proliferation activity of cells was detected by CCK-8 assay. Cells were exposed to Ang II at a concentration of 100 nmol/L or gradient concentrations of ZYZ-384, incubated for 48 h, and then analyzed for their proliferation or inhibitory effects. (A) Effect of ZYZ-384 on the viability of human HMEC-1 cells. (B) Effect of ZYZ-384 on the viability of mouse SVEC4-10 cells. (C) The stimulating proliferation effect of ZYZ-384 on human HMEC-1 cells. (D) The stimulating proliferation effect of ZYZ-384 on mouse SVEC4-10 cells. (E) SA-β-Gal staining. Aged cells exhibit a blue response. (#P < 0.05 vs. Ctrl group, # #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Ang II group, * *P < 0.01 vs. Ang II group).

Full size image

Analysis of the growth-promoting ability of ZYZ-384 on cells

The EdU (5-ethynyl-2’-deoxyuridine) staining technique is a highly sensitive method for detecting cellular proliferation11. We employed this approach to examine the impact of ZYZ-384 on the proliferation of human HMEC-1 or mouse SVEC4-10 cells. Upon addition of Ang II to both cell types, the actively dividing cells promptly entered an inhibited state. However, when ZYZ-384, Ang II, and cells were co-cultured under suppressed conditions, the cells immediately transitioned into a proliferation state (Fig. 2).

Fig. 2
figure 2

The proliferation effect of ZYZ-384 on human HMEC-1 or mouse SVEC4-10 cells is evidenced by the presence of bright green fluorescence. (A) (C) The impact of ZYZ-384 on the proliferation of human HMEC-1 cells and their fluorescence activity. (B) (D) The impact of ZYZ-384 on the proliferation of mouse SVEC4-10 cells and their fluorescence activity. (#P < 0.05 vs. Ctrl group, # #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Ang II group, * *P < 0.01 vs. Ang II group).

Full size image

The impact of ZYZ-384 on markers of aging

SMYD3 can promote the ubiquitination of p53 and is a crucial component in the aging process, interacting with p21 and p1612,13. Modulating the activity of p53, p21, and p16 can induce cell cycle arrest and re-entry14. Treatment of both cell types with Ang II led to cellular senescence, accompanied by a significant increase level of markers for p21, p16, and p53 in these cells. Gradual addition of ZYZ-384 effectively suppressed the expression of these markers (Figs. 3 and 4).

Fig. 3
figure 3

(A-D) The impact of ZYZ-384 on the expression levels of aging markers p21, p16, and p53 in human HMEC-1 cells, as well as the fluorescence activity of the cells. (#P < 0.05 vs. Ctrl group, # #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Ang II group, * *P < 0.01 vs. Ang II group).

Full size image
Fig. 4
figure 4

(A-D) The effect of ZYZ-384 on the expression levels of aging markers p21, p16, and p53 in mouse SVEC4-10 cells, as well as the fluorescence activity of the cells. (# #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Ang II group, * *P < 0.01 vs. Ang II group).

Full size image

Pathological alterations of ZYZ-384 in subacute senescence animal model

D-galactose, as an aldohexose, maintains a high concentration in the body and can be catalyzed by galactose oxidase to convert into aldoses and hydroperoxides, thus producing reactive oxygen species (ROS)15. Meanwhile, reactive oxygen and nitrogen species (RONS) can affect the body’s antioxidant capacity, and continuous oxidative stress can lead to the accumulation of aging, eventually leading to multi-organ dysfunction and disease16. Cellular experiments have confirmed that ZYZ-384 can delay cell aging. We induced subacute aging phenotypes in mice by injecting them with D-galactose, and then observed the effects of ZYZ-384 on the subacute aging model. Hematoxylin and eosin staining was made on the hippocampus, kidney, spleen, liver and heart of mice to observe their structural changes. In the control group, the nucleus of neurons in the hippocampus was deeply stained and basophilic, with no vacuolization observed in the neurons. The glomerulus in the kidney has a smooth and complete structure, normal size and shape, as well as an obvious cystic cavity structure. The spleen exhibits a higher count of newly formed germinal centers, indicating robust lymphocyte division and proliferation, as well as an augmented presence of red pulp. The liver comprises a substantial number of cells, characterized by basophilic nuclei. Notably, there is an absence of fibrosis, inflammatory cell infiltration, or hepatocyte degeneration. In the heart, the muscle fibers are arranged neatly, the nucleus is centrally located, and there is no inflammation or fibrosis. In the model group, the nucleus staining of brain neuron cells became pale, and intracellular vacuolation occurred. The mesangial margin of the glomerulus appeared irregular and showed a reduced shape. The basement membrane of the renal tubules was thickened and accompanied by atrophy. The number of new germinal centers in the spleen decreased, accompanied by white pulp dysplasia and a reduction in the red pulp area. The cell nucleus vacuolated and the number of hepatocytes decreased. The cardiomyocytes were arranged normally with gaps, and the nuclei were located in the middle of the cells. There was no inflammation present. Compared to the model group, the nuclei of hippocampal neurons in each dose group of ZYZ-384 were deeply stained, and only a few cells showed vacuolation. The mesangium in the kidney is intact, slightly larger in size, and exhibits an evident cystic cavity structure. A small number of germinal centers remained in the spleen, while the area of white pulp hyperplasia decreased and the area of red pulp expanded. The number of hepatocytes in the liver increased, while the number of vacuolated cells decreased. The myocardial cells exhibited normal arrangement, with centrally located nuclei and absence of inflammatory signs. The myocardial changes were not evident before and after the experiment (Fig. 5).

Fig. 5
figure 5

Hematoxylin-eosin staining images of a subacute aging mouse model formed by subcutaneous injection of D-galactose are shown from left to right, depicting the brain, kidney, spleen, liver, and heart.

Full size image

Regulatory effects of ZYZ-384 on aging markers in subacute aging animal model

The markers of aging included p16, p21, and Rb17,18. The expression levels of p16, p21, and Rb varied in the hippocampus, kidney, spleen, liver, and heart of mice following subcutaneous injection of D-galactose. After D-galactose stimulation, the level of p16 in these organs was up-regulated. However, upon intervention with varying concentrations of ZYZ-384, the level of p16 in these organs gradually decreased. The expression of p21 is up-regulated in these organs with the stimulation of D-galactose, but it is not obvious in the heart. After being stimulated with different concentrations of ZYZ-384, high doses of ZYZ-384 can significantly reduce the expression of p21 in the hippocampus. There was no change observed of p21 expression in the heart. Different concentrations of ZYZ-384 significantly suppressed p21 expression in the liver, spleen, and kidney. After induction of senescence by D-galactose, the expression of Rb was up-regulated in the hippocampus, kidney, spleen and liver in animal models. Different concentrations of ZYZ-384 to animal models resulted in down-regulation of Rb expression in the hippocampus, liver and kidney, while the high dose stimulated a decrease of Rb expression in the spleen. There was no change of Rb levels in the heart (Figs. 6, 7 and 8).

Fig. 6
figure 6

Effect of ZYZ-384 on the organ aging marker p16 in subacute senescence animal model. (A) Immunohistochemical staining images. (B) IHC score. The score was based on the intensity of staining and the proportion of positive cells. (#P < 0.05 vs. Ctrl group, # #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Model group, * *P < 0.01 vs. Model group).

Full size image
Fig. 7
figure 7

Effect of ZYZ-384 on the organ aging marker p21 in subacute senescence animal model. (A) Immunohistochemical staining images. (B) IHC score. (# #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Model group, * *P < 0.01 vs. Model group).

Full size image
Fig. 8
figure 8

Effect of ZYZ-384 on the organ aging marker Rb in subacute senescence animal model. (A) Immunohistochemical staining images. (B) IHC score. (#P < 0.05 vs. Ctrl group, # #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Model group, * *P < 0.01 vs. Model group).

Full size image

Differential metabolite analysis of ZYZ-384 in regulating subacute aging in an animal model

We used metabolomic methods to analyze the differences in plasma metabolites among the control group, the model group, and the high-dose group treated with ZYZ-384. The peaks were extracted from all experimental samples and quality control (QC) samples for principal component analysis, which can reflect both the overall differences between samples within each group and the degree of variation between samples within a group (Fig. 9A and B). The experimental results demonstrate that QC samples cluster closely in both positive and negative ion modes, indicating the robustness and good repeatability of the experiment. By performing structural identification of metabolites in biological samples, a total of 264 metabolites were identified between the control group and the model group, and 223 metabolites were identified between the model group and ZYZ-384 group. A total of 113 overlapping metabolites demonstrating significant differences were identified across the control, model, and ZYZ-384 experimental groups through multivariate analysis, employing statistical thresholds of P < 0.05 and OPLS-DA variable importance projection (VIP) scores exceeding 1.0. (Fig. 9C). Stratified cluster heat map was created for the top 50 species, highlighting metabolites with significant differences (Fig. 9D). The differential metabolites were analyzed for enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. 9E and F). We found that the phenylalanine metabolic pathway was an important pathway to distinguish the control group, the model group and the ZYZ-384 group. The three differential metabolites associated with the phenylalanine pathway are L-aspartate-semialdehyde, 3-hydroxybenzoic acid, and phenylpyruvic acid.

Fig. 9
figure 9

(A) (B) PCA analysis of the positive and negative ion models in the samples shows good dispersion with no overlap, indicating reliable data. (C) Venn diagram. (D) Heat map showing potential biomarker levels in different groups. Red indicates up-regulation, green indicates down-regulation. (E) (F) KEGG enrichment analysis in different groups. A represents ZYZ-384.

Full size image

Effects of ZYZ-384 on biochemical markers in plasma

Figure 10 shows that malondialdehyde (MDA), interleukin-1β (IL-1β), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), alanine transaminase (ALT), aspartate transaminase (AST), globulin (GLOB), creatinine (CREA), creatine kinase (CK), and blood urea nitrogen (BUN) were up-regulated, superoxide dismutase (SOD), albumin (ALB) and catalase (CAT) were down-regulated, and total cholesterol (TC) and triglyceride (TG) did not change in the D-galactose subacute aging animal model. When animal models were stimulated with different concentrations of ZYZ-384, MDA, IL-1β, IL-6, MCP, ALT, AST, GLOB, CREA, CK, and BUN were down-regulated, ALB, SOD and CAT were up-regulated, and TC and TG were not changed.

Fig. 10
figure 10

Effects of different concentrations of ZYZ-384 on SOD, MDA, IL-1β, IL-6, MCP-1, ALT, AST, GLOB, CREA, CK, BUN, Cat, TC and TG in plasma of a D-galactose induced subacute aging animal model. (# #P < 0.01 vs. Ctrl group; *P < 0.05 vs. Model group, * *P < 0.01 vs. Model group).

Full size image

Ethology and morphological analysis of ZYZ-384 in a natural aging model

Compared with the natural aging group, the rod turning time of mice in the ZYZ-384 groups significantly increased, the running persistence time significantly extended, and the search time on the platform significantly shortened. Compared with the ZYZ-384 H group, the ZYZ-384 H + siRNA SMYD3 group significantly prolongs the rod turning time and running time, enhancing their endurance. At the same time, it significantly shortens the platform search time. However, there is no difference between the ZYZ-384 H group and the siRNA SMYD3 group. In comparison to the siRNA SMYD3 group, the ZYZ-384 H + siRNA SMYD3 group significantly prolongs the running time and shortens the platform search time (Fig. 11A). After staining frozen sections with SA-β-Gal, aged tissues turned blue. Figure 11B clearly shows that there are obvious blue reactions in naturally aging animals’ hippocampus, kidney, spleen, and liver. When animals were interfered with by ZYZ-384 or siRNA SMYD3 treatment, the blue response of their hippocampus, kidney, spleen and liver became significantly blurred. Interestingly enough, no corresponding change in color was observed in heart tissue.

Fig. 11
figure 11

Effect of ZYZ-384 on ethology and morphology of natural aging model. (A) The ethology results, from left to right, include the rotating rod test, running experiment, and Barnes Maze experiment. (B) The SA-β-Gal staining of frozen sections of hippocampus, kidney, spleen, liver and heart. (*P < 0.05 vs. Model group; #P < 0.05 vs. ZYZ-384 H group; △P < 0.01 vs. siRNA SMYD3 group).

Full size image

Effects of ZYZ-384 on SMYD3 and H3K4me3 proteins

Our previous researches have confirmed that elevated levels of histone H3 lysine 4 (H3K4) methyltransferase SMYD3 protein and elevated H3K4me3 modification occur in Ang II-induced endothelial cell senescence, and that SMYD3 knockdown blocks endothelial cell senescence13. Figures 12 and 13 clearly demonstrate that the small molecule compound ZYZ-384, synthesized by us, significantly inhibits the levels of SMYD3 and H3K4me3 in the hippocampus, kidney, spleen, and liver, which is completely opposite to the performance of the model group. However, it was puzzling that there was no regulation observed in the expression of SMYD3 and H3K4me3 in the heart.

Fig. 12
figure 12

Effects of ZYZ-384 on SMYD3 protein in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (* *P < 0.01 vs. Model group; #P < 0.05 vs. ZYZ-384 H group; # #P < 0.01 vs. ZYZ-384 H group; △P < 0.05 vs. siRNA SMYD3 group; △ △P < 0.01 vs. siRNA SMYD3 group).

Full size image
Fig. 13
figure 13

Effects of ZYZ-384 on H3K4me3 protein in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (* *P < 0.01 vs. Model group; #P < 0.05 vs. ZYZ-384 H group; # #P < 0.01 vs. ZYZ-384 H group; △P < 0.05 vs. siRNA SMYD3 group; △ △P < 0.01 vs. siRNA SMYD3 group).

Full size image

Effect of ZYZ-384 on Hsp 90 and NF-κB p65

Heat shock protein 90 (Hsp 90), as one of the molecular chaperones for many proteins, can assist in protein synthesis and maintain homeostasis, as well as aid in cell signal transduction. Inhibiting the over-expression of Hsp 90 helps prolong the lifespan of C. elegans19. As a molecular chaperone for SMYD3, the Hsp 90 can promote chromatin binding of SMYD320. Additionally, Hsp 90 can activate IKK-α to induce downstream inflammatory signal NF-κB, which is involved in aging SASP21. Therefore, we preliminary verified the regulatory effect of ZYZ-384 on the SMYD3/Hsp 90/NF-κB signaling pathway. Hsp 90 and NF-κB p65 are over-expressed in the brain, kidney, spleen, and liver of naturally aging animal models. However, their levels are significantly inhibited by ZYZ-384 intervention. The ZYZ-384 H + siRNA SMYD3 group performed the best. It should be noted that Hsp 90 and NF-κB p 65 in the heart remain unaffected (Figs. 14 and 15).

Fig. 14
figure 14

Effect of ZYZ-384 on Hsp90 in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (* *P < 0.01 vs. Model group; #P < 0.05 vs. ZYZ-384 H group; # #P < 0.01 vs. ZYZ-384 H group; ∆P < 0.05 vs. siRNA SMYD3 group; ∆∆P < 0.01 vs. siRNA SMYD3 group).

Full size image
Fig. 15
figure 15

Effect of ZYZ-384 on NF-κB p65 in animal models of natural aging. (A) Immunohistochemical staining images. (B) IHC score. (* *P < 0.01 vs. Model group; # #P < 0.01 vs. ZYZ-384 H group; ∆∆P < 0.01 vs. siRNA SMYD3 group).

Full size image

Discussion

Chromatin, which consists of DNA and proteins within the nucleus, undergoes dynamic chemical alterations known as post-translational modifications on histone proteins. These modifications, including prominent examples like acetylation, methylation, phosphorylation, and ubiquitination, occur on specific amino acid residues within histone tails and cores. They function as a complex epigenetic regulatory code, critically modulating chromatin accessibility and gene expression while leaving the underlying DNA sequence unaltered. Among these mechanisms, histone methylation stands out as an especially intricate and functionally diverse process. This modification is specifically catalyzed by histone methyltransferases and reversed by demethylases. Methylation primarily targets lysine residues on histones H3 and H4, with extensively studied sites including H3K4, H3K9, H3K27, and H4K20. The functional outcomes of methylation are highly context-dependent22,23. Specifically, methylation at H3K4 is typically associated with transcriptional activation and euchromatin formation, whereas methylation at H3K9 or H3K27 is often linked to gene silencing and heterochromatin formation. This complex regulatory system substantially enhances the functional diversity of chromatin. Furthermore, our previous research has demonstrated that the lysine methyltransferase SMYD3 serves as a critical regulator within this network by catalyzing the upregulation of H3K4me3 trimethylation, a histone mark strongly correlated with active transcription and, notably, the promotion of cellular aging processes. Given the central role of SMYD3 in driving aging through the elevation of H3K4me3 levels, we developed a targeted therapeutic strategy. Specifically, we carried out an extensive screening campaign to identify promising lead compounds and subsequently synthesized a novel, highly potent small-molecule inhibitor, ZYZ-384, which was rationally designed to selectively target SMYD3. This compound served as our primary tool to investigate the feasibility of SMYD3 inhibition as an anti-aging intervention. Our preliminary validation strategy employed complementary in vitro and in vivo models. We induced senescence in both human and mouse endothelial cells using Angiotensin II (Ang II). Utilizing this model, we assessed the anti-senescence potential of ZYZ-384 through multiple assays. SA-β-Gal staining assessed the proportion of senescent cells, revealing that ZYZ-384 significantly reduced senescent cell burden. EdU staining measured DNA synthesis as an indicator of cell proliferation, demonstrating that ZYZ-384 treatment effectively enhanced proliferative capacity. Immunofluorescence staining was used to evaluate the expression levels of key intracellular senescence markers, including p16, p21, and γH2AX. The results confirmed that ZYZ-384 significantly reduced the expression of these markers. Taken together, these in vitro findings provide evidence that ZYZ-384 effectively delays cellular senescence and promotes cell proliferation. We utilized a well-established D-galactose-induced subacute aging model in mice. Within this in vivo context, we systematically evaluated the systemic effects of ZYZ-384. Our results demonstrated that ZYZ-384 significantly attenuated the expression of aging-related markers across multiple organs, including the brain, kidney, spleen, and liver. Crucially, ZYZ-384 demonstrated potent inhibitory effects on the SASP, significantly reducing levels of pro-inflammatory cytokines and other inflammatory mediators in the blood. The inhibitor effectively downregulated SMYD3 expression in the brain, kidney, spleen, and liver. ZYZ-384 mitigated D-galactose-induced liver and kidney dysfunction, suggesting functional organ protection. Applying metabolomics technology, we identified that ZYZ-384 profoundly impacts metabolic pathways, with the phenylalanine pathway emerging as the most significantly altered in the subacute aging model. This finding intriguingly aligns with previous research by Czibik et al., linking perturbed phenylalanine metabolism to aging phenotypes24. To confirm on-target activity, we utilized SMYD3 knockdown, which phenocopied key effects of ZYZ-384 treatment. This provided preliminary evidence that ZYZ-384 exerts its anti-aging effects in vivo by specifically targeting SMYD3 and subsequently modulating the H3K4me3/Hsp90/NF-κB signaling pathway, a cascade critically involved in inflammation and senescence. While our findings are promising, several limitations warrant consideration. The absence of substantial changes observed specifically in the heart during aging experiments may be attributed to factors such as strain variability among animals, inadequate experimental duration, or intrinsic tissue-specific sensitivities to aging stimuli or interventions. Aging is an extraordinarily intricate and multifactorial process governed by interconnected hallmarks, including cellular senescence, genomic instability, telomere shortening, epigenetic modifications, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, stem cell exhaustion, and altered intercellular communication25. Furthermore, it involves systemic changes like body composition shifts, tissue degeneration, and energy metabolism dysregulation26. While animal and cell models provide essential, controllable systems for mechanistic insight, they inherently simplify the human condition. Human aging involves vastly more complex genetic, environmental, and lifestyle factors. Extrapolating results directly to humans requires caution. Although achieving true “reversal” of the aged state remains a profound and arguably currently intractable scientific challenge effectively requiring the repair of extensive accumulated damage and restoration of a youthful homeostatic equilibrium our research demonstrates that modulating the aging trajectory is feasible. Strategies like caloric restriction, exercise, and pharmacological interventions hold significant potential to slow aging processes. This rationale underpinned our choice of 13-month-old mice, which are equivalent to humans entering their 40 s, a critical transition period in aging, for natural aging observations. Advancing this field necessitates extensive further exploration. Future work should focus on refining SMYD3 inhibitors, investigating long-term efficacy and safety, exploring combinatorial approaches targeting multiple aging pathways, utilizing more complex models such as human organoids, diverse genetic backgrounds, and ultimately translating these findings towards promoting healthier human aging.

Conclusion

The small molecule compound ZYZ-384, synthesized by us, can significantly inhibit SMYD3, promote the proliferation of human and mouse endothelial cells, and alleviate the changes in aging markers in animal organs caused by subacute senescence induced by D-galactose. By regulating the SMYD3/H3K4me3/Hsp 90/NF-κB signaling pathway in natural aging model animals, these results provide valuable insights for the development of anti-aging agents.